Allergens are antigenic molecules inducing allergic responses and production of IgE antibodies in humans. They are used both for diagnosis and for treatment of allergy i.e. allergen immunotherapy, the latter in the form of allergen vaccines. Allergenic source materials to which humans are exposed, such as foods, pollens or mite faecal particles occur naturally as complex mixture(s) of major and minor allergens. Major allergens are allergens to which a majority of patients, who are allergic to the source, react. It appears, however, that any protein is a potential allergen, since still more and more minor allergens are identified as knowledge increases.
Due to the complexity of the allergen sources, the amino acid sequences of several allergens were first deduced from cDNA-derived nucleotide sequences. Cloning of genes encoding allergens revealed that most allergens are heterogeneous and that they occur as mixtures of isoallergens and variants. Amino acid sequence alignment of homologous allergens and isoallergens demonstrated that they can be identified by and/or divided into constant and variable region sequences. Constant region amino acid sequences are unique to the species however variable region amino acid sequences are unique to each of the isoallergens.
Conventional allergen specific immunotherapy and diagnosis are currently performed by use of standardized natural allergen extracts which are further formulated to allergen vaccines. These aqueous vaccines are based on allergenic natural source materials, such as tree and grass pollen, dust mite cultures and animal hair and dander particles. The composition of these natural source materials are known to vary considerably depending on time and place of collection of the allergenic source materials. Commercial allergen vaccines can furthermore be formulated to mixtures of allergens using various species.
Knowledge of the composition of the extracts and the content of essential allergens is a prerequisite for reproducibility, safety and efficacy of the final product. A major challenge in the manufacture of allergen vaccines is standardisation, i.e. securing a constant potency from batch to batch. Since the raw material is of natural origin, variation is considerable and needs to be controlled by scientifically based measures. The composition of the extract should ideally reflect the composition of the water soluble components of the allergenic source material as it is extracted on the mucosal surface of the airways and presented to the human immune system. All extracts, however, contain several allergens contributing to the total IgE-binding in different combinations for individual patients. Ideally, therefore, all components need to be controlled both qualitatively and quantitatively, but with the current technology this is not practically possible.
Standardisation is currently performed in many different ways, since each manufacturer has company specific standardisation procedures. Standardisation is performed by techniques such as SDS-PAGE, isoelectric focusing in addition to a variety of immunoelectrophoretic (QIE) and ELISA techniques using mono- and/or polyclonal antibodies and radio allergosorbent (RAST) or related techniques. An optimal batch-to-batch standardisation, such as the SQ-standardisation procedure, is essentially a three step procedure: 1) securing optimal composition and constant ratios between all components by semi-quantitative immuno-electrophoretic techniques, 2) determining major allergen components by quantitative immunoelectrophoresis, and 3) adjusting the overall IgE-binding potency as determined in Magic Lite® assays. In Europe, all standardisation is currently performed relative to an in-house company specific reference preparation, whereas in the U.S., the FDA issues standards to be used by all manufacturers. All quantitative aspects of these currently used techniques are dependent on antibodies as reagents and as such vulnerable to change over time.
Absolute quantification of specific vaccine components in complex mixtures of allergen is not straightforward and has yet not been established as a sensitive, routine high-through-put technique.
Also in the food industry routine high-through-put techniques for reliable detection and quantification of food allergens is necessary. Nuts may be found as a hidden part of a food because of accidental cross-contamination during manufacturing. Companies producing similar foods with and without e.g. nuts may have difficulty in cleaning production equipment in between making the different types of foods. Traces of previously produced foods such as nuts can remain on the equipment. The first batches of foods made without nuts that go through the same equipment will likely contain traces of nuts. Foods that may cause allergic reaction due to cross-contamination of nuts or peanuts are e.g. chocolate, candies, cookies, desserts, sweets, donuts, cereal, milkshakes, granola bars, muesli, pies, muffins, ice cream, barbecue sauce. Cows' milk may cause an allergic reaction to small amounts of milk protein from dairy products, from cows' milk, formula based on cows' milk or baby foods containing protein from milk. To avoid contamination of milk protein during production of baby foods or baby formula to milk allergic children a detection and quantification method for milk allergens is therefore needed. Reliable detection and quantification methods for food allergens are necessary in order to ensure compliance with food labelling and to improve consumer protection. Physicochemical methods e.g. mass spectrometry, as well as immunological methods have been described. The usual criteria of sensitivity, specificity, reproducibility, precision and accuracy have to be fulfilled. Still, there remain problems of cross-reactivity, of matrix effects and of food processing. Biological activity may remain when the protein is denatured.
Biological mass spectrometry (MS) was first employed to assess molecular weight and identity of proteins and peptides. In recent years, advances in mass spectrometry have resulted in techniques which can be used for quantification of a variety of biomolecules from complex mixtures such as plasma, cell and tissue samples. Earlier quantification techniques have established only relative quantification of proteins whereas the more recent techniques assess absolute quantities of molecules of interest. The rapid development of quantification techniques is mainly due to progress in the field of proteomics, particularly in applications distinguishing, for instance, healthy and diseased states and identification of marker molecules for several diseases such as cancer, rheumatoid arthritis and Alzheimers disease. The major advantage of these quantification techniques by MS is the high sensitivity of the techniques ranging from 300 amol to 300 fmol of samples.
WO 2004/070352 discloses a method for quantification of peptides relative to an internal standard using isobaric labelling reagents or sets of isobaric labelling reagents.
U.S. Pat. No. 6,872,575 discloses a method for identification of one or more proteins in complex sample mixtures without purifying the protein or obtaining its composite peptide signature.
U.S. Pat. No. 6,864,089 discloses a method for quantification using differential isotopic labelling of peptide or protein samples.
Other methods for quantification of proteins using MS techniques are e.g. the AQUA technique using internal calibration peptides synthesized with incorporated stable isotopes (13C, 15N) to mimic native peptides formed by enzymatic digestion using e.g., trypsin (Stemmann O et al. Cell 2001; 107(6):715-26, Gerber S A et al. Proc Natl Acad Sci USA 2003; 100(12):6940-5).
Another method is the ICPL (Isotope Coded Protein Labelling) method described by Kellermann et al, Proteomics 5, 4-15, using e.g 12C/13C6-Nicotinic acid-succinimide as ICPL label.
Mass spectrometry was first introduced to the field of allergy research to characterize natural allergens including post translational modifications such as glycosylation patterns. It was further employed to characterize recombinant isoallergens and/or variants many of which have been expressed in various expression systems such as Escherichia Coli, Pichia Pastoris and Baculovirus expression systems.
Johannes et al, J Allergy Clin Immunol, Vol. 110, No. 1 (2002), pages 131-138 describes the use of MS to study the actual expression of allergen isoforms identified by PCR cloning and in Swoboda et al., J. Biol Chem, Vol 270, No. 6 (1995), pages 2607-2613, liquid chromatography, MS and cDNA cloning is used to analyze isoforms of the major birch pollen allergen, Bet v 1.
There still is a need for sensitive method, however, by which active components such as allergens from the same species or different species, and/or isoallergens e.g. in a vaccine may be quantified. A method using MS techniques and the species specific and allergen specific amino acid sequences provides a very sensitive method by which the content of specific allergens or groups of allergens (isoallergens or homologous allergens) may be quantified. The method according to the invention is useful e.g. in a release assay in order to ensure a safe and accurate amount of allergen during production of a vaccine, in the final product, and also during the various stages of storage of active ingredients and/or products. The method would also be beneficial in development of second generation allergen vaccines e.g. using recombinant allergens as active ingredients. Such a method would make it possible to optimize active ingredients in second generation allergen vaccines based on the knowledge and/or composition of the current vaccines.